Cell division is essential for proliferation of bacteria, and appears to rely on a molecular machine positioned at the site of division. Despite extensive knowledge of the major proteins involved in this fundamental cellular process, very little is understood about how these proteins are targeted, how they assemble into a complex, and how they radically alter normal cell wall growth in order to synthesize the division septum. The long term goals of this project are to elucidate the molecular mechanisms behind the targeting and assembly of cell division proteins, using Escherichia coli as a model system and taking advantage of new, powerful cytological methods for bacteria. A key universal cell division protein, FtsZ, self-assembles into a polymeric ring (the Z-ring) at the surface of the inner membrane at the division site and recruits other proteins, including FtsA, ZipA, and a group of integral membrane proteins, all of which help to complete the septum. The first aim of this proposal is to gain a molecular understanding of the recruitment of FtsA and ZipA by FtsZ. One way that this will be addressed is by mutagenesis of a region of FtsZ that is involved in FtsZ-FtsA and FtsZ-ZipA interactions in order to obtain proteins defective in interaction. Once such mutants are isolated, selection for intragenic and extragenic suppressors may reveal residue-specific contacts. Novel fluorescent in situ protein interaction assays will be used to screen for protein-protein interactions, followed by biochemical tests for direct interaction. The second aim will investigate the role of FtsA in cell division. Enzymatic and structural properties of purified FtsA will be characterized. In addition, the ability of FtsA to interact with FtsZ, ZipA, and downstream cell division proteins in the membrane will be tested. The final aims of the project are to understand how the Z-ring gets targeted to its exact cellular address. The nucleoid appears to negatively influence Z-ring assembly, and it is possible that nucleoid segregation releases a negative signal to allow precise Z-ring positioning. Studies of Z-ring assembly in mutants defective in nucleoid structure and chromosome replication will be carried out to determine the relationship between the nucleoid and the Z-ring. Finally, the Min system, which consists of MinC, D, and E, is an important regulator of Z- ring assembly and positioning. The relationship between the Min system and FtsZ will be addressed in detail both biochemically and genetically. The results of this proposed research are expected to provide a more complete understanding of the molecular mechanisms of cell division, and should facilitate the isolation of new and better antimicrobial drug targets.